The present invention relates to microbial fermentation processes that utilize genetically-modified bacterial or yeast cells (microbial) to produce a pyrimidine deoxyribonucleoside (PdN) in recoverable quantities.
Pyrimidine deoxyribonucleosides, such as thymidine (TdR) and deoxyuridine (UdR), are commercially useful starting compounds for the production of antiviral compounds comprising synthetic analogs of pyrimidines, such as azidothymidine (AZT) or azidodeoxyuridine (AZdU). For example, thymidine and deoxyuridine (UdR) are currently produced by organic synthesis utilizing a multi-step process which is very costly. Thus the high cost of thymidine has contributed to the high cost of antiviral therapeutics, e.g., the resulting high price of the drug AZT. There have not been any alternative methods, however, for obtaining thymidine or other PdNs suitable for use in producing antiviral therapeutics such as AZT. Thus, there is a longstanding and urgent need for a less expensive method for the production of commercially useful quantities of PdNs.
Exemplary of PdNs are thymidine and deoxyuridine, which are composed of the bases thymine and uracil, respectively, covalently attached at their 1-nitrogen to the 1'-carbon of the sugar 2-deoxyribose. These nucleosides normally exist in cells as mono-, di- or trinucleotides. Thymidine functions primarily as a component of DNA. In most cells of microorganisms and eukaryotic organisms, PdNs are products of the de novo pyrimidine biosynthetic pathway.
Analysis of PdN biosynthetic pathways. In order to provide an alternative, less expensive method for PdN production, the analysis of the metabolic pathways of PdN de novo synthesis might provide useful information as to possible cell mutants that could produce a PdN by the use of microbial fermentors or cell culture bioreactors. Pyrimidines, pyrimidine nucleosides, and pyrimidine nucleotides are synthesized from aspartic acid and carbamoyl phosphate (derived from glutamine and CO.sub.2) by way of a multi-step pathway, which is shown in FIG. 1. See O'Donovan & Neuhard, Bacteriol. Rev. 34:278-343 (1970), the contents of which are herein incorporated by reference. The enzymes of the pathway, indicated in FIG. 1 by the E. coli gene symbols, are listed below:
pyrA or carAB--Carbamoyl phosphate synthase (EC 6.3.5.5) PA0 pyrBI--Aspartate transcarbamoylase (EC 2.1.3.2) PA0 pyrC--Dihydroorotase (EC 3.5.2.3) PA0 pyrD--Dihydroorote oxidase (EC 1.3.3.1) PA0 pyrE--Orotate phosphoribosyltransferase (EC 2.4.2.10) PA0 pyrF--Orotidine 5'-phosphate decarboxylase (EC 4.1.1.23) PA0 pyrG--Cytidine triphosphate (CTP) synthase (EC 6.3.4.2) PA0 pyrH--Nucleoside-phosphate kinase (EC 2.7.4.4) PA0 ndk--Nucleoside diphosphate kinase (EC 2.7.4.6) PA0 nrd--Ribonucleoside diphosphate reductase (EC 1.17.4.1) PA0 dcd--Deoxycytidine triphosphate (dCTP)-deaminase (EC 3.5.4.13) PA0 dut--Deoxyuridine triphosphate nucleotide hydrolase (dUTPase) (EC 3.6.1.23) PA0 thyA--Thymidylate synthase (EC 2.1.1.45).
The endproducts of the pathway can be generally defined as uridine triphosphate (UTP), cytidine triphosphate (CTP), deoxycytidine triphosphate (dCTP) and thymidine triphosphate (TTP), all of which function as building blocks for RNA or DNA as well as for other cellular components. Because of the large number of endproducts, energy requirements and enzymatic steps in this pathway, de novo biosynthesis of pyrimidines has many regulated steps. See, e.g., Neuhard, In THE MOLECULAR BIOLOGY OF BACTERIAL GROWTH, Jones & Bartlett, Boston (1985) at pages 173-184.
Regulation of the pyrimidine de novo biosynthetic pathway. The enzymatic steps of the pyrimidine de novo biosynthetic pathway are regulated in vivo by (A) feedback inhibition of key enzymes by the concentration of the endproducts or other metabolites (either alone or in concert); and (B) repression and/or attenuation of enzyme synthesis. Beckwith et al, J. Mol. Biol. 5:618-634 (1962); Potvin et al, J. Bacteriol. 123:604-615 (1975); Roland et al, J. Bacteriol. 163:991-999 (1985). This pathway in bacteria has been studied in detail. Neuhard & Nygaard, In ESCHERICHIA COLI AND SALMONELLA TYPHIMURIUM, CELLULAR AND MOLECULAR BIOLOGY, American Society for Microbiology, Washington, D.C. (1987), at pages 445-473.
According to Neuhard, id., the key regulatory enzymes of pyrimidine biosynthesis are carbamoyl phosphate synthase (pyrA or carAB), aspartate transcarbamoylase (pyrBI), CTP synthase (pyrG) and deoxycytidine triphosphate deaminase (dcd). Carbamoyl phosphate synthase activity is inhibited by uridine nucleotides and its synthesis is cumulatively repressed by arginine and uracil. Regulation by arginine is necessary because carbamoyl phosphate is an intermediate in the biosynthesis of arginine as well as pyrimidines. In E. coli a specific repressor protein, argR, has been identified. In Bacillus subtilis two carbamoyl phosphate synthase isoenzymes have been identified (Paulus & Switzer, J. Bacteriol. 137:82-91 [1978]). One is specifically regulated by arginine and the other by pyrimidines (primarily uracil derivatives).
Aspartate transcarbamoylase (AT) catalyzes the first committed step in pyrimidine biosynthesis and is therefore subject to metabolic regulation by pyrimidines. The enzyme AT consists of two types of subunits, the catalytic subunits (pyrB), and the regulatory subunits (pyrI). The activity of AT is subject to allosteric inhibition by CTP, one of the endproducts of the pathway, and activation by adenosine triphosphate (ATP), whose concentration is dependent upon of the energy state of the cell. The level of AT present in the cell is apparently regulated by CTP and UTP. The encoding gene and associated regulatory regions have been cloned and their DNA sequences determined. See Landick & Yanofsky, In ESCHERICHIA COLI AND SALMONELLA TYPHIMURIUM, CELLULAR AND MOLECULAR BIOLOGY, 1276-1301; Roland & Powell, supra: and Turnbough et al, Proc. Nat'l Acad. Sci. USA 80:368-72 (1983).
Constitutive E. coli mutants have been described in which the level of AT was elevated about 30-fold over normally repressed levels. However, there have also been experiments described in which starvation of an E. coli pyrimidine auxotroph for pyrimidines resulted in about a 1000-fold increase in the level of the AT enzyme (Shepherdson & Pardee, J. Biol. Chem. 235:3233-37 [1960]). Thus, the range of enzyme levels is very great and depends on the levels of the end products in the cell and, under normal, repressed conditions, the enzyme is present at much lower levels than are possible under fully-deregulated conditions.
The regulation of AT in B. subtilis differs from that in E. coli and S. typhimurium in that no in vitro activation or inhibition of enzyme activity has been demonstrated (Bethell & Jones, Arch. Biochem. Biophys. 134:352-65 [1969]). There is, however, regulation of enzyme synthesis by uracil, probably by an attenuation mechanism similar to that proposed for the other organisms. Lerner & Switzer, J. Biol. Chem. 261:11156-65 (1986).
The third key enzyme is CTP synthase (pyrG) which catalyzes the amination of UTP to form CTP. The enzyme from E. coli is subject to complex allosteric regulation. In vitro studies demonstrate stimulation by GTP, ATP, and UTP. The mechanism of its genetic regulation is not known. The fourth key enzyme is dCTP deaminase, which catalyzes the deamination of dCTP to dUTP. The substrate dCTP is a branch point compound since it is either incorporated into DNA or used as the precursor for TTP synthesis. The enzyme dCTP deaminase is subject to feedback inhibition by TTP. In B. subtilis dCTP or dCDP is converted to dCMP, which is then utilized as the substrate for dCMP deaminase, producing dUMP as the product. However, dUMP does not accumulate in the cell because it is immediately converted to TMP by the enzyme thymidylate synthase. Munch-Petersen, ed., METABOLISM OF NUCLEOTIDES, NUCLEOSIDES AND NUCLEOBASES IN MICROORGANISMS, Academic Press, 149-201 (1983). The enzyme dCMP deaminase is subject to allosteric activation by dCTP and inhibition by TTP.
Another enzyme, ribonucleoside diphosphate reductase (nrd), plays a key role in the biosynthesis of pyrimidine deoxyribonucleotides and is subject to complex regulation. Thelander & Reichard, Ann. Rev. Biochem 48:133-58 (1979). This enzyme catalyzes the reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates and acts on both purine and pyrimidine nucleotides. However, most inhibition studies have been performed on purified enzyme and may not truly reflect the situation in vivo, since the enzyme is normally membrane-associated. The level of enzyme activity generally seems to be a function of the total nucleotide concentration in the cell, as well as the rate of DNA synthesis.
In E. coli and S. typhimurium, the pyrimidine biosynthetic genes are scattered throughout the chromosome and do not constitute an operon (Beckwith, supra; Neuhard, In THE MOLECULAR BIOLOGY OF BACTERIAL GROWTH, supra, (1985) at pages 173-84) except for the subunits of carbamoyl phosphate synthase (carA and carB) and the subunits of aspartate transcarbamoylase (pyrB and pyrI). As mentioned above, their expression appears to be regulated at the level of transcription by an attenuation mechanism, which is dependent upon the intracellular concentrations of CTP and UTP. Any situation in which the levels of CTP or UTP are low will result in increased expression of these enzymes.
To date no specific repressor protein acting on the pyrimidine biosynthetic genes has been discovered and coordinate expression of the genes has not been seen. In B. subtilis., the genes pyrA, pyrBI, pyrC, pyrD, pyrE, and pyrF are located close to one another on the chromosome (Lerner et al, J. Bacteriol. 169:2202-06 (1987), the contents of which are herein incorporated by reference) and are expressed at very low levels when the organism is grown in the presence of uracil (Potvin et al, J. Bacteriol. 123:604-15 [1975]), but the evidence so far also points to regulation by an attenuation mechanism (Lerner, supra, 1986). There is no clear evidence for an operon type of gene organization, nor for the existence of a repressor protein.
Lack of accumulation of pyrimidine deoxyribonucleosides. The enzyme reactions which involve a representative PdN (thymidine) are shown in FIG. 2. In a wild type cell growing under non-starvation conditions, thymidine is not present or exists at very low levels because the precursor deoxyuridine monophosphate (dUMP) is converted directly to thymidine monophosphate (TMP) and then to thymidine diphosphate (TDP) and thymidine triphosphate (TTP). Since thymidine is present primarily in the form of TTP, a building block for DNA, it is normally utilized as rapidly as it is synthesized and does not accumulate, due to regulated biosynthesis. For the same reasons, deoxyuridine also is not normally found in cells. Existing pyrimidine metabolic pathways therefore normally preclude the accumulation of excess deoxyuridine (UdR), dTMP or thymidine (TdR).
Although alternative metabolic mechanisms can result in the synthesis of thymidine, the thymidine is either degraded or used by the cell for synthesis of thymidine nucleotides, thus preventing the accumulation of thymidine. For example, the action of non-specific phosphatases on TMP to produce thymidine does not lead to the accumulation of thymidine because any thymidine produced is either degraded rapidly to thymine and deoxyribose-1-phosphate by the action of thymidine phosphorylase, the product of the deoA gene (Munch-Peterson, supra, 203-58), or phosphorylated by thymidine kinase to produce TMP. In B. subtilis, there appears to be only one pyrimidine nucleoside phosphorylase which can use either thymidine or uridine as the nucleoside substrate. This enzyme also functions in the reverse manner, allowing the incorporation of exogenous thymine into thymidine. Many organisms can also take up thymidine and convert it to TMP by the action of thymidine kinase. As a consequence, thymidine does not accumulate via the metabolic pathways existing in organisms. Deoxyuridine produced from dUMP by non-specific phosphatases also serves as a substrate for thymidine kinase, yielding dUMP, or as a substrate for thymidine phosphorylase, yielding uracil and deoxyribose 1-phosphate.
Although there are several examples of production of nucleosides, pyrimidine-like compounds, and related products by fermentation, there is no suggestion that PdN production could be obtained in a similar manner, since in these cases mutations of existing pathways provided accumulation of the desired pyrimidine or pyrimidine ribonucleoside, but did not suggest or provide for the accumulation of pyrimidine deoxyribonucleosides. See, e.g., Konishi, Chem. Abs. 70:18917j (1969); Nakayama, Chem. Abs. 84:119951k (1976); and Nakayama, Chem. Abs. 89:74214g, 88:119441b, 88:19442c (1978).
Indeed, such a mutation, or other metabolic mechanisms described above, could not provide for commercially useful amounts of thymidine or deoxyuridine to accumulate because of the above-mentioned removal of thymidine (or deoxyuridine) by either TMP (or dUMP) synthesis or degradation to thymine (or uracil) and deoxyribose-1-phosphate. Because the regulatory mechanisms for the pyrimidine biosynthetic genes appear to be primarily attenuation mechanisms, with each gene individually regulated, it would not be expected or predictable that mutants highly de-repressed for the whole pathway could be generated, resulting in the accumulation of specific PdNs in recoverable amounts. Also, it has not been possible heretofore to exploit existing metabolic pathways in any organism to accomplish practical PdN production by rapid and specific irreversible hydrolysis of TMP to thymidine or dUMP to deoxyuridine, as the former is formed in vivo. This has followed from the fact that the equilibrium of the thymidine kinase reaction is unfavorable for TdR or UdR production.
Accordingly, no approach has been available that provided or suggested a solution to the problem of producing commercially useful amounts of PdN at relatively low cost. Moreover, known means of modification of existing metabolic pathways have not provided for the accumulation of recoverable amounts of PdNs such as TdR during microbial fermentation, due to extensive regulation and unfavorable reaction equilibria in relevant synthetic steps. Thus a need exists to solve the problem of commercial production of recoverable quantities of a PdN.